Reference intervals for thiopurine S-methyltransferase activity in red blood cells using 6-thioguanine as substrate and rapid non-extraction liquid chromatography.

BACKGROUND: Although widely used, thiopurine drugs have a narrow therapeutic index and treatment can result in life-threatening toxicity, the basis being pharmacogenetic variation in thiopurine metabolism by thiopurine S-methyltransferase (TPMT). We recently developed a modified phenotyping assay to determine TPMT activity in red blood cells. Here we describe improvements to the method and establish reference intervals in a large prospective study. METHODS: A modified enzyme assay for TPMT activity is reported. It uses 6-thioguanine as substrate with heat treatment of the incubate to stop the reaction and precipitate protein prior to high-performance liquid chromatographic (HPLC) analysis. Measurement of the reaction product, 6-methylthioguanine (6-MTG), uses HPLC with fluorimetric detection. RESULTS: The assay shows excellent characteristics, with clear discrimination of patients who are deficient in TPMT activity (< 5 nmol 6-MTG per g Hb per h) from heterozygotes (5-24 nmol 6-MTG per g Hb per h) and patients with normal activity (>25 nmol 6-MTG per g Hb per h). CONCLUSION: A modified TPMT assay is described which is suited for routine analysis in a regional centre. The method overcomes the need for extraction and has speeded up the chromatographic determination of 6-MTG, enabling large numbers of samples to be analysed. A prospective study of 1000 individuals has established the distribution of TPMT activity using the assay.

An example of the healing powers of a human being.

A 60-year-old man was referred as an emergency with a 3 month history of left sided abdominal pain and weight loss. He had no other medical problems and took no medications. An endoscopy was performed. This demonstrated a normal oesophagus and stomach and a fleshy mass in the first part of the duodenum with surrounding slough. As this was presumed to be malignant, biopsies were taken. Computed tomography (CT) scan of the abdomen was performed which showed a large, complex, loculated intra-abdominal collection containing air. Results from duodenal biology showed the presence of ulcer slough and liver tissue. The patient was diagnosed with a perforated duodenal ulcer, which had occurred some months previously, and which had eroded into the liver. He was observed and treated with intravenous antibiotics. The patient was discharged on day 14. Follow-up CT scan at 6 weeks showed complete resolution of the collection.

Ethnic variation of thiopurine S-methyltransferase activity: a large, prospective population study.

UNLABELLED: The use of azathioprine (and its metabolite mercaptopurine) is limited by toxicity, especially myelosuppression, which is related to activity of the enzyme thiopurine S-methyltransferase (TPMT). TPMT activity varies between individuals and is considered deficient in one in 300 cases. AIMS & METHODS: We identified TPMT activity within an ethnically diverse population of patients attending an inner-city hospital phlebotomy service. A total of 1000 subjects were recruited and analyzed with respect to age, sex and ethnicity. RESULTS: Samples were analyzed from 456 Caucasians, 342 South Asians and 180 Afro-Caribbeans. Six subjects had deficient TPMT activity (0.6%: four women, two men; four Caucasians, one Afro-Caribbean, one South Asian). TPMT activity (nmol 6-methylthioguanine (6-MTG)/gHb/h) ranged 0-76 (median [interquartile range]: 33 [28-39]). Enzyme activity was lower in Afro-Caribbeans (30 [25-37.5]) than Caucasians (34 [29-40]) and South Asians (33 [29-38]), which was significant after adjustment for age and sex (p < 0.0001). Activity was lower in women (p = 0.022), especially South Asian females (n = 194; 32 [28-36]), compared with (35 [30-40]) in men (n = 148; p = 0.002). CONCLUSIONS: A higher prevalence of TPMT deficiency was recorded than in previous studies. Afro-Caribbeans have lower activity than Caucasians and South Asians. TPMT enzyme activity was lower among females, especially in South Asians.